Interest is building to consider somatic gene therapy as an approach to heritable diseases of the osteoblast such as osteogenesis imperfecta (OI). Significant problems which may be unique to bone and other connective tissue cells need serious consideration before a cogent strategy can be formulated. Specifically, it needs to be determined whether there is a bone stem cell that can be targeted which will yield a progenitor pool of differentiated osteoblasts expressing the correcting gene. Five fundamental questions will be asked by this application, utilizing transgenic donor mice bearing marker genes which can indicate the source and the differentiation state of cells derived from a manipulated stem cell: (1) do renewing osteoblasts naturally arise from a circulating stem cell, a tissue resident stem cell, or both?; (2) what is the molecular and cellular basis of apparently normal bone strength in patients who are somatic mosaics for a mutation causing OI? For stem cell therapy to be successful, engineered cells will need to enter a natural pool of circulating cells to populate OI bone, replace the function of the OI cells and ameliorate disease severity. These issues will be answered with allophenic mice derived from embryo donors bearing different transgenes; (3) even if stem cells do not normally circulate, can stem cells be made to populate bone and yield differentiated osteoblasts? MSC will be expanded in vitro and used in a heterotopic and intramedullary assay of bone formation and stem cell number. A mouse engineered with the TK gene, controlled by the COL1A1 promoter, will be used to assess the ability of transplanted MSC to engraft bone and participate in bone formation; (4) and (5) can MSC be manipulated with retroviral vectors containing either ubiquitous, or a COL1A1 promoter and still maintain its ability to home to, and make bone? This project represents a fusion of three principal investigators, bringing different, but interacting disciplines to the complex question of feasibility of MSCs as the cellular ingredient in an overall strategy of somatic gene therapy of bone.